\documentclass{bioinfo}
\copyrightyear{2011}
\pubyear{2011}

\begin{document}
\firstpage{1}

\title[Phenylketonuria]{Phenylketonuria: Mahjong on Phenylalanine Hydroxylase}
\author[Meier and Pfeiffenberger]{Erik Pfeiffenberger and Markus Meier\footnote{correspondence should be addressed to the email address: meiermark@cip.ifi.lmu.de}}
\address{Department for Bioinformatics and Computational Biology, Faculty for Informatics, Boltzmannstrasse 3, Garching 85748, Germany}

\history{Received on September 30, 2011}

\editor{Associate Editor: Edda Kloppman, Andrea Schafferhans, Mark Offman}

\maketitle

\begin{abstract}

\section{Motivation:} Phenylketonuria (PKU) is a metabolic disease which reduces the depletion of phenylalanine and thus causes toxic concentrations of this amino acid in blood with the effect that affected individuals suffer from e.g. mental retardations. PKU has a prevalence of up to 1 in 4000 in Turkey. The most common reason for PKU is one or more harmful mutations in a gene called PAH (phenylalanine hydroxylase). The in silico analysis done in this paper tries to shed  light in the mechanisms and function of PAH based cases of PKU. 


\section{Results:} The result section discusses the implications to protein function and stability of the four mutations R168Q, R261Q, P281L and R408W in the protein phenylalanine hydroxylase. These are the most common mutations observed in patients suffering from phenylketonuria. The results for each mutation were entirely produced by in silico analysis of the phenylalanine hydroxylase protein. This analysis was done sequence based and structure based. Sequence based analysis is focusing on the conservation and physicochemical properties of each mutation. The structure based analysis focuses on the alterations to the H-bond network and their implications to the protein stability. 

\section{Availability:} All results are available from http://goo.gl/LlEHs.

\section{Contact:} \href{meiermark@cip.ifi.lmu.de}{meiermark@cip.ifi.lmu.de}
\end{abstract}

\section{Introduction}
%why is PKU important to be researched: incidence
Phenylketonuria (PKU) has been first discovered and described by Ivar Asbj\o rn F\o lling in 1934 as a metabolic disease which has strong effects on mental abilities of affected individuals, e.g. reduced intelligence or hyperactivity, if untreated in newborns (\citealp{folling1934ausscheidung}). The frequency of reported PKU cases is highly heterogeneous among different populations. In Turkey 1 in 4000 births show PKU with classical symptoms which is the highest observed incidence rate in the world. This high rate is mainly caused by high consanguinity within the population (\citealp{blau2010phenylketonuria} and \citealp{ozalp2001newborn}). High incidence rates are also observed in regions of northern and eastern Europe and Italy (\citealp{DiLella}). On the other hand countries such as Finland, China and Thailand are examples for low incidence countries. They have prevalence rates of 1 in 100,000 births in Finland (\citealp{Guldberg01121995}), 1 in 100,500 births in some parts of China (\citealp{jiang2003survey} and \citealp{zhan2009neonatal}) and  less than 1 in 200,000 births in Thailand (\citealp{pangkanon2009detection}). However, due to PKU's high prevalence in most countries it is reasonable to put further effort into research in order to be able to develop new treatments which may be more beneficial for affected individuals. 

\subsection*{Diagnosis}
%diagnosis
In the last decades diagnosis of phenylketonuria shifted away from a clinical, symptom orientated, diagnosis to a biochemical diagnosis. Due to neonatal screening newborns can be diagnosed early in life before symptoms develop after 10 to 14 days. This is done by measuring the concentration of phenylalanine in blood. A standard method known as the "heel prick" test is normally applied to all newborn infants for this purpose. This test simply takes blood from the heel of the infant which is then taken to test against a range of genetic diseases for example cretinism, cystic fibrosis and phenylketonuria of course (\citealp{van2010phenylketonuria} and \citealp{guthrie1963simple}). 

\subsection*{PKU's protagonists and their roles}
%what is the source of PKU: defect in PAH...
PKU is caused by a severe defect of the phenylalanine abolishing pathway which leads to toxic concentrations of phenylalanine. 99\% of PKU associated mutations map to the protein phenylalanine hydroxylase (PAH) (\citealp{Erlandsen}). However, phenylalanine hydroxylase requires three helper molecules for the process which are O2, Fe$^{2+}$ and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). BH4 is synthesized from guaninethreephosphate (GTP) in a three step process which requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). During the hydroxylating process of phenylalanine to tyrosine the molecule BH4 is consumed and has to be recycled in order to be reused again in another hydroxylating process. This recycling process is catalyzed by the two enzymes pterin-4a-carbinolamindehydratase (PCD) and the NADH-dependent dihydropteridine reductase (DHPR) (\citealp{blau2010phenylketonuria}). 
The remaining 1\% of the PKU associated mutations map to proteins involved in the biosynthesis and regeneration of the cofactor of PAH (\citealp{Erlandsen}).

\subsection*{Macroscopic disease effect}
PAH is responsible for the disposal of about 75\% of the dietry phenylalanine (\citealp{Erlandsen}), thus it is the major pathway to reduce the concentration of phenylalanine. The reduced activity of phenylalanine hydroxylase in patients with phenylketonuria leads to harmful concentrations of phenylalanine. A normal concentration of phenylalanine in the human blood ranges from 50 to 110 $\mu$mol/L. Values above that can be interpreted as toxic. Depending on the concentration of phenylalanine in the blood different categories are applied. Individuals with values from 120 to 600 $\mu$mol/L are classified as having mild hyperphenylalaninemia (HPA), 600 to 1200 $\mu$mol/L is classified as mild phenylketonuria and individuals with concentrations above 1200 $\mu$mol/L are classified as having the classical phenylketonuria (\citealp{blau2010phenylketonuria}).
The toxicity of phenylalanine can be explained by its relation to the blood-brain barrier. Phenylalanine is a large neutral amino acid. There is a transporter across the blood-brain barrier for these kind of acids, known as the large neutral amino acid transporter. Large neutral amino acids compete for the transportation by this enzyme. If phenylalanine is over-represented other amino acids are missing in the brain which is especially critical during brain development (\citealp{blau2010phenylketonuria}).
%REMARK is there also anothter reference? i think blau mentioned this only shortly - denke es ist logisch nachvollziehbar auch ohne konkretere quelle


\subsection*{Treatment}
%treatment
The most common strategy to reduce phenylalanine concentration in affected individuals is by reducing the intake of phenylalanine rich food. This has to be done from the very first day when phenylketonuria is diagnosed in infants to avoid the commonly known symptoms of phenylketonuria. These dietary products are mainly low protein products (\citealp{blau2010phenylketonuria}). 
%shortened to stick to the 6 pages limitation
Affected individuals have to avoid food like e.g. meats, fish, eggs, standard bread, most cheeses, nuts, and seeds. In addition there is also a need to avoid drinks which contain aspartame, flour and soya. Also beer or cream liqueurs are not recommended. Recommended food include potatoes, some vegetables, and most cereals products. However, even these products shall be eaten only in a restricted manner. In alternative to natural products the industry provides special low-protein food for affected individuals like e.g. low-protein bread and low-protein pasta (\citealp{blau2010phenylketonuria}). 
In addition to the well established diet therapy there is ongoing research on new therapies. One promising approach could be the injection of phenylalanine ammonia lyase to patients. This bacterial protein is able to catalyze a transformation of phenylalanine to trans-cinnamic acid and ammonia without any cofactor requirements (\citealp{macdonald2007modern}). So far, this therapy was successful in mouse models of phenylketonuria (\citealp{sarkissian2008preclinical}). Another approach to fight phenylketonuria could be gene therapy. This approach tries to introduce a vector of a functional PAH gene to the DNA. If successful, individuals will be able to express their own phenylalanine hydroxylase protein (\citealp{jung2008protective}, \citealp{rebuffat2010comparison} and \citealp{ding2008correction}).

\section{Phenylalanine hydroxylase}
%general informations
Phenylalanine hydroxylase (PAH) is an iron-containing enzyme localized in the cytosol. PAH is one of the pterin-dependent amino acid hydroxylases, hence PAH requires the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) in order to catalyze the conversion of phenylalanine to tyrosine.
A properly functional phenylalanine hydroxylase protein realizes the transformation from phenylalanine to tyrosine by hydroxylating the substrate phenylalanine. More precisely, it adds an OH group to the fourth position of the 6-carbon aromatic ring of phenylalanine, thus resulting in a tyrosine (\citealp{blau2010phenylketonuria}). 

\subsection*{Inheritance}
%inheritance
Individuals who suffer from phenlyketonuria which is caused by a dysfunction of PAH require two mutated alleles of the PAH gene, thus it is inherited in a autosomal recessive fashion (\citealp{james2006andrews}). They have to be mutated in a way that the resulting protein product is severe dysfunctional in its ability to catalyze the transformation from phenylalanine to tyrosine. This is only possible when both healthy parents carry one dysfunctional allele on their chromosome 12.  Their offspring will then have a 25\% chance to be affected by phenylketonuria because these individuals inherited both dysfunctional PAH alleles from their parents. Furthermore, there is only a 25\% for their offspring to be a non-carrier of a dysfunctional allele and a 50\% chance to inherit exactly one dysfunctional allele of the PAH gene. 

\subsection*{Gene and sequence}
%gene & sequence
The PAH gene is located on the long arm of the autosomal chromosome 12 between positions 22 and 24.2 in humans. The precise location is defined from base pairs 103,232,103 to 103,311,380 which results in a total length of 79,277 bps on the chromosome . This gene consists of 13 exons and 12 introns, after the introns of the pre-mature mRNA are spliced away a length of only 2,681 bps is left on the transcript (\citealp{ensemblpah} and \citealp{scriver2007pah}). This means only 3.38\% of the original gene size is left on the mature mRNA. However, the full length of a functional phenylalanine hydroxylase protein is 452 residues after translation.

\subsection*{Protein reaction}
%protein reaction
PAH catalyzes the conversion of phenylalanine to tyrosine. This reaction can be separated in three steps (\citealp{fitzpatrick2003}):

\begin{enumerate}
\item formation of a Fe(II)-O-O-BH4 bridge.
\item heterolytic cleavage of the O-O bond to yield the ferryl oxo hydroxylating intermediate Fe(IV)=O
\item attack on Fe(IV)=O to hydroxylate phenylalanine substrate to tyrosine
\end{enumerate}

The exact mechanism behind especially the first step is still highly discussed.


\subsection*{Protein structure}
%protein structure
\begin{figure}[!tpb]
\centerline{\includegraphics[width=0.5\textwidth]{full.jpeg}}
\caption{The structure of the homotetramere complex of PAH. source: \citealp{Erlandsen}}\label{fig:06}
\end{figure}

The structure of PAH is very well reviewed. The following shall be a summary of the results in order to get a better understanding of the effect of mutations on PAH. The eukaryotic PAH exists in an equilibrium between homotetrameric and homodimeric forms (figure \ref{fig:06} shows the homotetrameric form) and consists of the following three domains:.

\begin{figure}[!tpb]
\centerline{\includegraphics[width=0.5\textwidth]{full_dom.jpeg}}
\caption{Visualization of the domain architecture of PAH. Yellow: catalytic domain. Blue: regulatory domain. Green: tetramerization domain. source:  \citealp{Erlandsen}}\label{fig:05}
\end{figure}

\begin{enumerate}
\item the regulatory N-terminal domain (residues 1 to 142)
\item the catalytic domain (residues 143 to 410)
\item the C-terminal oligomerization domain (residues 411 to 453)
\end{enumerate}

Figure \ref{fig:05} shows the domain architecture of PAH. PAH is regulated by a kind of allosteric activation which means that the substrate phenylalanine needs to be present at a certain concentration and after a short delay the enzyme starts to hydroxylate phenylalanine. This behavior can be explained by the regulatory N-terminal domain, which ranges from residues 1 to 142 in PAH. The regulatory nature of this domain is caused by its structural flexibility (\citealp{Erlandsen}).

Hydrogen and deuterium exchange analysis show that the interface between the regulatory and the catalytic domain becomes more exposed to the solvent by the binding of phenylalanine. This can be explained by a global altering of the conformation of the enzyme. This change contains an exposing of the active site to the solvent.

This argumentation is supported by kinetic studies. These studies show an initially low rate of tyrosine formation for full-length PAH. This lag time is not observed, for a truncated PAH lacking the N-terminal domain or if the full-length enzyme is pre-incubated with phenylalanine. The deletion of the N-terminal domain results in the elimination of the lag time and an increasing of the affinity for phenylalanine by nearly two-fold (\citealp{fitzpatrick2010}, \citealp{fitzpatrick2011} and \citealp{Kobe1999}).

An additional source of regulation is SER16 in PAH. The phosphorylation of SER16 does not alter the enzyme conformation but does reduce the concentration of phenylalanine required for allosteric activation (\citealp{Kobe1999}).


\subsection*{Catalytic domain}
The catalytic domain ranges from residue 143 to 410 in PAH. The pocket, representing the active site, is primarily lined by hydrophobic residues. This pocket contains three glutamic acid residues, two histidines, and a tyrosine, which are critical for pterin- (residues 264 to 290) and iron-binding (H285, H290, E330). There are two different models, which describe the events after the binding of phenylalanine.
The first suggests, that all coordinated water molecules are forced out of the active site and BH4 becomes directly coordinated to the iron (\citealp{Olsson2011}).
The second suggests, that the iron shifts from a six- to a five-coordinated state by removing a single water molecule and opening a binding site for oxygen. At the same time BH4 is shifted towards the iron atom (\citealp{Bassan2003}). However, the discrepancy between the two models could not be solved yet. 

\subsection*{C-terminal oligomerization domain}
The C-terminal oligomerization domain ranges from residue 411 to 453 in PAH. The procaryotic PAH is monomeric. Although, eukaryotic PAH exists in an equilibrium between homotetrameric and homodimeric forms. The dimerization interface is composed of symmetry-related loops that link identical monomers. The overlapping C-terminal tetramerization domain mediates the association of conformationally distinct dimers that are characterized by a different relative orientation of the catalytic and tetramerization domains. A domain swapping mechanism has been proposed to mediate formation of the tetramer from dimers shifting equilibrium towards the tetrameric form. In this mechanism C-terminal alpha-helices mutually alter their conformation around a flexible C-terminal five-residue hinge region to form a coiled-coil structure (\citealp{fitzpatrick2003}, \citealp{Flatmark1999} and \citealp{Bjorgo2001}).

Both the homodimeric and homotetrameric forms of PAH are catalytically active. But the two exhibit different kinetics and regulation. In addition to reduced catalytic efficiency, the dimer does not display allosteric activation by phenylalanine. This suggests that phenylalanine allosterically regulates PAH by influencing the dimer-dimer interaction (\citealp{Bjorgo2001}).

\section{Approach}
There are several experimental structures of PAH, but none of them captures the whole protein (see \citealp{listpahstruc} for a list of available structures).
Most crystallographers were interested in the catalytic domain. Therefore they used a trimmed protein. Especially the flexible N-terminal and C-terminal domains were removed, because such regions are prone to cause errors in the resolution of the experimental structure. For structure dependent methods we used the X-Ray structure with the description "Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II) in Complex with Tetrahydrobiopterin" and the PDB identifier 1J8U. This structure has a high resolution of 1.50 \AA and captures residues from 103 to 427 (\citealp{andersen2001high} and \citealp{pdb1j8u}).

In order to predict the effect of several mutations, we applied several in silico methods to PAH. The results of these methods give us more insight in the protein. The static structure of 1J8U itself is very simple, but it is still too complex to be interpreted manually.

The secondary structure gives us hints, which parts of the protein are more defined than others. Mutations in very defined environments could disturb the enzyme's function dramatically. The secondary structure of PAH was assigned with the help of the structure 1J8U by employing DSSP (\citealp{kabsch1983dictionary}). However, since 1J8U does not solve the whole structure of PAH we had to employ PSI-PRED (\citealp{jones1999protein}) to predict the structure of the unsolved regions.

The flexibility of a protein in certain regions indicates possible movements of the protein, which are necessary for the function.
The flexible regions of 1J8U are estimated by the b-factor of the alpha carbons. %Different possible motions of the structure are calculated in normal mode analysis.

For each mutation several characteristics of the amino acid substitutions are regarded to predict their impact on the enzyme.
The physicochemical properties of the two amino acids are compared. 
The effect on the structure without changing the backbone conformation was estimated with SCWRL. 
Therefore the native amino acid was replaced with a fitting rotamer of the mutation amino acid by SCWRL, then the native and mutated H-bonds are compared. 
There are some annotations on functional sites of PAH. The mutations are regarded with respect to these known functional sites.

\begin{methods}
\section{Methods}

We employed several tools for different kinds of analysis to receive a comprehensive understanding of PAH and the effect of mutations to it. The following sections describe the employed methods shortly. 

\subsection*{Structure analysis}

We employed two kinds of methods to analyze the structure:

\paragraph*{Sequence Based:} We used only one sequence based method. PSI-PRED predicts the secondary structure of an amino acid sequence. For this purpose it produces a sequence profile with PSI-BLAST. A two-level neural network is fed with windows of the profile and predicts the secondary structure (\citealp{jones1999protein}).

\paragraph*{Structure Based:} The structure 1J8U was analyzed with several methods. DSSP estimates the secondary structure of an experimental structure by regarding the distances of certain residues. If the distance between two residues is below a defined threshold a H-bond is assumed between fitting residues. The secondary structure elements are then distinguished by H-bond patterns which are unique for each type of secondary structure (\citealp{kabsch1983dictionary}). 


\subsection*{Side chain placement}
The position of side chains of our mutations was computed by SCWRL which is a side-chain placement tool. It takes an experimental structure and some specified amino acids in the structure. It tries to place an optimal side chain conformation of a rotamer library for this specified amino acids with respect to an energy function (\citealp{canutescu2003graph}).


\subsection*{Conservation analysis}
We applied a multiple sequence alignments (MSA) with homologous sequences in mammalians in order to identify conserved regions in PAH. This MSA was facilitated with t-coffee 3D. This alignment tools does not align the sequences directly but uses their structural information to generate templates and align these first in order to compute the actual sequence alignment (\citealp{poirot20043dcoffee}). Furthermore we compared the scores of the mutations to a point accepted mutation matrix (PAM) (\citealp{dayhoff1978model}), a block substitution matrix (BLOSUM) (\citealp{henikoff1992amino}) and a position specific scoring matrix (PSSM) generated from a PSI-BLAST (\citealp{altschul1997gapped}).

\subsection*{Energy calculations of mutants}
In order to determine the effect of mutations on overall protein stability we used the programs FoldX (\citealp{schymkowitz2005foldx}) and Groningen Machine for Chemical Simulations (GROMACS) (\citealp{van2005gromacs}) to calculate their forcefields. 
%TODO add subsection which shortly describes foldX, minimise, etc...

\subsection*{H-Bond distance calculation}
The distance calculations of our H-bond network in wild type and mutants has been performed with Pymol, a molecular graphic tool (\citealp{pymol}).

\subsection*{Flexibility analysis}

We performed our molecular dynamics simulation which gave us insights into the motion and flexibility of PAH with the simulation suit called GROMACS (\citealp{van2005gromacs}).


%NMA describes a method to calculate possible movements of a structure.

\end{methods}

\section{Results and Discussion}

%\begin{table*}[!t]
%\processtable{The analyzed mutations with the annotated effect from HGMD and a summary of the corresponding analysis \label{Tab:01}}
%{\begin{tabular}{llp{13cm}}\toprule
%Mutation & Associated Disease & Summary of Analysis\\\midrule
%I65T & Phenylketonuria q & row1\\
%R71H & Hyperphenylalaninaemia & row2\\
%R158Q & Phenylketonuria & row3\\
%R261Q & Phenylketonuria & R261 has a H-bond to T238; H-bond is removed by mutation; possible shift of the helix next to R261; helix next to R261 contains the BH4 binding site; changes in the distance of BH4 and the iron are likely to destroy the catalytic reaction mechanism \\
%T266A & Neutral & row3\\
%P275S & Hyperphenylalaninaemia & row3\\
%T278N & Hyperphenylalaninaemia & row3\\
%P281L & Phenylketonuria & P281 is localized at the end of a helix, which is part of the catalytic site; mutation of the helix-breaker probably causes major changes in the binding surface, especially the binding sites of the iron\\
%G312D & Neutral & no interactions in the native structure; solvent exposed loop not associated to the catalytic cleft; expected to have no effect\\
%R408W & Phenylketonuria & concentration of interactions to the catalytic and the oligomerization domain; mutation caused clashes in the prediction of SCWRL; mutation probably disturbs a proper oligomerization \\\botrule
%\end{tabular}}{}%This is a footnote}
%\end{table*}

\begin{table}[!t]
\processtable{The predicted H-bonds in the native structure and in the mutated structures created by SCWRL for each mutation\label{Tab:02}}
{\begin{tabular}{lllllll}\toprule
\multicolumn{3}{c}{From} & \multicolumn{3}{|c|}{To} & \\
AA & SC/BB & Atom & AA & SC/BB & Atom & Distance (\AA)\\\midrule
R158 & SC & N &E280 & SC & O & 2.9\\
R158 & SC & N & E280 & SC & O & 2.8\\
R158 & SC & N & E141 & SC & O & 3.6\\ 
R158 & SC & N & Y154 & SC & O & 3\\
R158 & BB & N & Y154 & BB & O & 2.9\\
R158 & BB & O & F161 & BB & N & 3.2\\
R158 & BB & O & A162 & BB & N & 2.9\\
Q158 & SC & N & Y154 & SC & O & 3.1\\
Q158 & SC & N & I269 & SC & O & 3.5\\
Q158 & BB & N & Y154 & BB & O & 2.9\\
Q158 & BB & O & F161 & BB & N & 3.2\\
Q158 & BB & O & A162 & BB & N & 2.9\\
R261 & SC & NH2 & T238 & BB & O & 2.3\\
R261 & BB & N & L258 & BB & O & 2.9\\
R261 & BB & O & R241 & BB & N & 2.9\\
Q261 & BB & N & L258 & BB & O & 2.9\\
Q261 & BB & O & R241 & BB & N & 2.9\\
%T266 & SC & O & E286 & SC & O & 3.1\\
%T266 & BB & N & E286 & SC & O & 2.8\\
%A266 & BB & N & E286 & SC & O & 2.8\\
%P275 & BB & O & R270 & SC & N & 2.9\\
%S275 & BB & O & R270 & SC & N & 3.1\\
%T278 & SC & O & E280 & BB & O & 2.7\\
%T278 & SC & O & E280 & BB & N & 3.3\\
%N278 & SC & O & E280 & BB & N & 3.4\\
P281 & BB & O & Y268 & SC & OH & 2.3\\
L281 & BB & O & Y268 & SC & OH & 2.3\\
R408 & SC & NH2 & L311 & BB & O & 2.8\\
R408 & SC & NH2 & A309 & BB & O & 3.0\\
R408 & SC & NH2 & L308 & BB & O & 2.9\\\botrule
\end{tabular}}{}%This is a footnote}
\end{table}

\begin{figure}[!tpb]
\centerline{\includegraphics[width=0.5\textwidth]{158_261_281_312_408.png}}
\caption{A visualization of the key players in the analysis of the mutations R158Q (a), R261Q (b), P281L (c) and R408W (d). Red: mutated residue; Dark-orange: bound iron; Magenta: BH4; Blue: oligomerization domain; Black: H-bonds between the oligomerization domain and the catalytic domain; Orange: residue 238} \label{fig:allmutations}
\end{figure}

\begin{figure}[!tpb]
\centerline{\includegraphics[width=0.5\textwidth]{rmsf.png}}
\caption{Shows the root mean square fluctuations of PAH as calculated by our molecular dynamics simulation for our WT.} \label{fig:rmsf}
\end{figure}


%\subsection*{Mapping point mutations}
%TODO point mutations
%In this paper we focus on mutations changing one amino acid of the human protein PAH and causing PKU.

%Show secondary structure with annotation of domains and functional/important sites with position numbers
%Do hydrophobicity plot... in pah are several regions of hydrophob residues
%\subsection*{I65T:}
%\subsection*{R71H:}

%TODO: shorten this section...
\subsection*{R158Q:}

\paragraph*{Sequence Based:}
The mutation from amino acid arginine to amino acid glutamine on position 158 leads to the effect that this residue is no longer positive charged which might have an influence on its neighbors in close proximity. In addition this mutation is located within an alpha-helix (see figure \ref{fig:allmutations}). The mutant glutamine is not know to be a helix breaker (helix breakers are proline or glycine for example) as well as it is not known to be one of the 5 most common alpha-helical amino acids (known as MALEK = methionine, alanine, leucine, uncharged glutamate, and lysine) (\citealp{nick1998helix}). Therefore, it is hard for us to tell whether this mutant has disturbing influence on the structure of the alpha-helix. Another interesting fact is that this mutation is not close to the catalytic site which has been identified to be the residues HIS285, HIS290, GLU330 and SER349. Hence, the possibility of direct influence of this mutation to the catalytic reaction can be excluded and other reasons such as reduction of overall protein stability are more likely to harm the proper function of PAH.
When analyzing the conservation of ARG158 with BLOSUM62 we got a score of 1 which is the second best score in this matrix. The worst score for a mutation from arginine to another amino acid is produced by either cysteine, isoleucine, phenylalanine or tryptophan with a score of -3. The best mutation score is produced by a mutation to lysine with a score of 2 (see \citealp{blossummatrix} for a reference of the complete matrix). Hence, we assume that our mutation from arginine to glutamine is quite accepted by BLOSUM62.
We got similar results for the PAM1 and PAM250 scoring matrices (see \citealp{pammatrix} for a reference of the complete matrix). For PAM1 we have got an score of 9 which is quite good with respect to a worst score of 0 and a best mutation score of 37. For PAM250 it is similar but not the same. Here we got a score of 5 with a worst score of 1 and the best mutation score of 9.
However, when we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation is not quite accepted in evolution (see \citealp{pssmmatrix} and \citealp{msahomo} for a reference of the complete PSSM and MSA). To be more precise: The mutant glutamine has been observed in 6\% of all cases for the position 158 whereas the WT arginine has a conservation of 50\% in our PSSM. The conservation of arginine is even more present in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 62/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this conservation is driven by some positive selection pressure which is suppressing a more liberal incorporation of other amino acids for this position. 

%\begin{figure}[!tpb]
%\centerline{\includegraphics[width=0.5\textwidth]{158_int.png}}
%\caption{A visualization of the key players in the analysis of the mutation R158Q. Red: residue 158; Dark-orange: bound iron; Magenta: BH4}\label{fig:158}
%\end{figure}

\paragraph*{Structure Based:}
When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that the mutated side chain of SCWRL points to the same direction as the lower part of the WT side chain. 
When analyzing the H-bond network we made the observation that our WT is strongly interconnected with other residues by H-bonds. To be more precise we found 7 H-bonds, 4 H-bonds are side chain to side chain H-bonds and the other 3 are backbone to backbone H-bonds (see table \ref{Tab:02}). This means that due to the mutation three side chain to side chain interactions got lost. When looking at the H-bonds in our mutant Q158 we find the three backbone to backbone H-bonds as in our WT and two side chain to side chain H-bonds. One of these H-bonds is the old  Q158 $\rightarrow$ Y154 H-bond which is preserved. Furthermore, we found a new H-bond which does not exists in our WT, which is Q158 $\rightarrow$ I269. However, the distance of this H-bond is 2.5 \AA. We assume that the threshold for a strong H-bond is 2.5 \AA so we suggest that this H-bond interaction is very weak here.
In our energy calculations with the methods foldX and gromacs we found indeed an increase of the energy which is visible from a positive delta value and supports our hypothesis of an loss of stability due to the mutation at position 158. In addition we found out that the mutation happens in a very rigid region of PAH. The root mean square fluctuation (RMSF) diagram in figure \ref{fig:rmsf} shows us that position 158 is located in a valley surrounded by more flexible regions. Thus, we may assume that due to the mutation (which had the effect that several H-bonds got lost) an increase of flexibility is caused in a region which is not favorable for it. 


\subsection*{R261Q:}

\paragraph*{Sequence Based:}
The mutation exchanges the positively charged arginine with the uncharged glutamine at the position 261. This mutation is located in a coil of the catalytic domain next to the helix, which contains the BH4 binding motive. The polarity of arginine could be necessary to form interactions, which are essential for the correct positioning of this helix. This functional importance of R261 is supported by our conservation analysis. We observed a conservation of 73/76 for the WT and a conversation of 0/76 for our mutant Q261 in our MSA of homologous mammalian sequences (see \citealp{msahomo} for the complete MSA).

\paragraph*{Structure Based:}

%\begin{figure}[!tpb]
%\centerline{\includegraphics[width=0.5\textwidth]{261_int.png}}
%\caption{A visualization of the key players in the analysis of the mutation R261Q. Red: residue 261; Orange: residue 238; Dark-orange: bound iron; Magenta: BH4}\label{fig:01}
%\end{figure}

The mutation seems to disrupt the H-bond to the threonine at position 238 in the predicted side chain conformation of SCWRL. This H-bond is probably necessary for the correct positioning of the helix next to the position 261 in the catalytic site, which contains the BH4 binding site (see figure \ref{fig:allmutations}b).

The change in polarity and the destroyed H-bond are probably causing a shift of the neighboring helix, which contains the BH4 binding motive. Such a shift would result in a changed location of the BH4 cofactor which will destroy the reaction mechanism in its last consequence.

%\subsection*{T266A:}

%\paragraph*{Sequence Based:}
%The mutation from amino acid threonine to amino acid alanine on position 266 leads to the minor effect that this residue is no longer polar but the rest of the physiochemical properties stay pretty much the same. Furthermore, this mutation is located within a loop which are known to be more liberal to mutations than secondary structure elements like alpha-helices and beta-strands. Another interesting fact is that this mutation is close to the catalytic site which has been identified to be the residues HIS 285, HIS 290, GLU 330 and SER 349. Hence, the possibility of a indirect or direct influence of this mutation to the catalytic reaction cannot be completely excluded.
%When analyzing the conversation of T266 with BLOSUM62 we got a score of 0 which is the second best score. The worst score for a mutation from threonine to another amino acid is produced by either glycine, histidine, phenylalanine, tryptophan or tyrosine with a score of -2. The best mutation score is produced by a mutation to serine with a score of 1. Hence, we assume that our mutation from threonine to alanine is quite accepted by BLOSUM62.
%We got similar results for the PAM1 and PAM250 scoring matrices. For PAM1 we have got an score of 32 which is quite good with respect to a worst score of 0 and a best mutation score of 38. For PAM250 it is similar but not the same. Here we got a score of 11 with a worst score of 0 and the best mutation score of 11.
%When we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation is can be told to be tolerated from a evolutionary point of view. To be more precise: The mutant alanine has been observed in 40\% of all cases for the position 266 whereas the WT arginine has a conservation of 40\% in our PSSM. The conservation of threonine is more present in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 48/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this position is not so much under positive selection pressure since we observed that both alleles can exists at this position (at least in our PSSM).

%\paragraph*{Structure Based:}
%When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that all three side chains point into the same direction. The rotation of pymol and SCWRL is the same. However, this is not surprising since alanine has no side chain.
%During our H-bond network analysis we found out that the WT, T266 is interconnected with other residues by H-bonds. To be more precise we found 2 H-bonds, the first H-bonds is a side chain to side chain H-bond and the second is a backbone to side chain H-bond. 
%Furthermore, we found out that one side chain to side chain interaction got lost. As expected our mutant A266 preserved the backbone to side chain h-bond from A266 to E286.
%In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value. However, this positive delta value occurred only for the calculations of gromacs and foldx. Minimise calculated a slightly negative delta value which means that our mutant is more stable than the WT. This seems to be a little awkward since we found out that several H-bonds get lost due to the mutation.

%\subsection*{P275S:}

%\paragraph*{Sequence Based:}
%The mutation from amino acid proline to amino acid serine on position 275 leads to the minor effect that this residue is now polar instead of unpolar but the rest of the physicochemical properties stay pretty much the same. This mutation is located within a turn which often incorporate small and tiny amino acids to facilitate the tight turn. So since serine is also a tiny residue this should have not so much an effect on the turn. Another interesting fact is that this mutation is not close to the catalytic site which has been identified to be the residues HIS 285, HIS 290, GLU 330 and SER 349.
%In BLOSUM62 we got a score of -1 which is the best score. The worst score for a mutation from proline to another amino acid is produced by either phenylalanine or tryptophan with a score of -4. The best mutation score is produced by a mutation to serine or threonine with a score of -1. Hence, we assume that our mutation from proline to serine is quite accepted by BLOSUM62.
%We got similar results for the PAM1 and PAM250 scoring matrices. For PAM1 we have got an score of 17 which is quite good with respect to a worst score of 0 and a best mutation score of 22. For PAM250 it is similar but not the same. Here we got a score of 9 with a worst score of 0 and the best mutation score of 11.
%When we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation is can be told to be tolerated from a evolutionary point of view. To be more precise: The mutant serine has been observed in 0\% of all cases for the position 266 whereas the WT arginine has a conservation of only 29\% in our PSSM. The conservation of proline is more present in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 48/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this position is not so much under positive selection pressure since we observed that proline has not a high conservation (at least in our PSSM).

%\paragraph*{Structure Based:}
%When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that all three side chains point into the same direction. Also the rotation of the mutatant side chain of pymol and SCWRL is the same.
%When analyzing the H-bond network we found out that the WT is interconnected with only one residue which is a backbone to side chain H-bond. 
%As expected this mutation is preserved in our mutant S275. However the distance of this H-bond increased from 2.9 angstrom in the WT to 3.1 angstrom in the mutant. So we assume that this might have a minor influence on the protein stability.
%In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value. However, this positive delta value occurred only for the calculations of gromacs and foldx. Minimise calculated a slightly negative delta value which means that our mutant is more stable than the WT.

%\subsection*{T278N:}

%\paragraph*{Sequence Based:}
%The mutation from amino acid threonine to amino acid asparagine on position 278 leads to the minor effect that this residue is no longer hydrophobic but the rest of the physicochemical properties stay pretty much the same. This mutation is located within a loop which are known to be more liberal to mutations than secondary structure elements like alpha-helices and beta-strands. Another interesting fact is that this mutation is close to the catalytic site which has been identified to be the residues HIS 285, HIS 290, GLU 330 and SER 349. Although, it is not close to one of these residues. Therefore, we assume that they have no direct influence but a indirect on the catalytic reaction.
%In a next step we analyzed the scores and conservation of this mutation to see how established scoring matrices like BLOSUM and PAM judge this mutation and how conserved this residue is in all mammalian homologous. In BLOSUM62 we got a score of 0 which is the second best score. The worst score for a mutation from threonine to another amino acid is produced with a score of -2. The best mutation score is produced by a mutation to serine or threonine with a score of 1. Hence, we assume that our mutation from threonine to asparagine is quite accepted by BLOSUM62.
%We got similar results for the PAM1 and PAM250 scoring matrices. For PAM1 we have got a score of 9 which is quite good with respect to a worst score of 0 and a best mutation score of 38. For PAM250 it is similar but not the same. Here we got a score of 4 with a worst score of 0 and the best mutation score of 11.
%When we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation can be told to be tolerated from a evolutionary point of view. To be more precise: The mutant asparagine has been observed in 2\% of all cases for the position 266 whereas the WT threonine has a conservation of only 33\% in our PSSM. The conservation of threonine is similar in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 34/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this position is not so much under positive selection pressure since we observed that threonine has not a high conservation.

%\paragraph*{Structure Based:}
%When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that the distinction between the mutated side chain position of pymol and SCWRL is that the mutated side chain of SCWRL is flipped to the empty C branch of the WT and pymols side chain is flipped to the CO branch of the WT. Hence, we may assume that they form different polar interactions.
%When analyzing the H-bond network we found out that the WT is interconnected with other residues by H-bonds. To be more precise we found two H-bonds, which are side chain to back bone H-bonds. We found that one side chain to backbone interaction got lost. In our mutant N278 we now find only one H-bond preserved which is the N278 -> E280 H -bond. So we may assume that we have a slightly decreased protein stability. Furthermore the hydrophobic threonine got interchanged with a non hydrophobic residue which also might have an influence.
%In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value.

\subsection*{P281L:}

\paragraph*{Sequence Based:}
This mutation removes the helix-breaker P from the end of a helix which makes it likely now that the broken helix extends and thus leads to changes in the backbone conformation. Furthermore we observed in our conservation analysis a conservation of 74/76 in WT and a conservation of 0/76 for the mutant L281 which is supportive for our hypothesis that P281 is needed to conserve PAH's conformation. 

\paragraph*{Structure Based:}

%\begin{figure}[!tpb]
%\centerline{\includegraphics[width=0.5\textwidth]{281_int.png}}
%\caption{A visualization of the key players in the analysis of the mutation P281L. Red: residue 281; Dark-orange: bound iron; Magenta: BH4}\label{fig:02}
%\end{figure}

In the prediction of SCWRL effects on the backbone conformation of this mutations will not be seen. But the structure enlightens the environment of mutation.
Residue 281 is in close proximity of the bound iron atom (see figure \ref{fig:allmutations}c). The predicted extension of the helix could cause major changes in the surface of the binding site of the iron, which is essential for the reaction. Therefore the mutation is probably limiting the catalytic activity of PAH.

%\subsection*{G312D:}

%\paragraph*{Sequence Based:}
%The unpolar and very small glycine is exchanged with the polar and small aperic acid. The position 312 is located in a coil. Depending on the interactions of the mutated residue and the location of the coil this could result in major changes.

%\paragraph*{Structure Based:}
%\begin{figure}[!tpb]
%\centerline{\includegraphics[width=0.5\textwidth]{312_int.png}}
%\caption{A visualization of the key players in the analysis of the mutation G312D. Red: residue 312; Dark-orange: bound iron; Magenta: BH4}\label{fig:03}
%\end{figure}
%The residue 312 has no interactions. The coil, which contains the mutation, is solvent exposed and does not seem to be crucial for the reaction. The mutation does not seem to influence any parts of the catalytic site or the binding sites. This mutation is probably not influencing the enzyme activity.

\subsection*{R408W:}

\paragraph*{Sequence Based:}
The positively charged arginine is exchanged with the aromatic, hydrophobic tryptophan. The mutation is located in a coil connecting the catalytic domain and the oligomerization domain. Depending on its interactions the mutation could influence the coordination of the two domains. This idea is supported by our conservation analysis too. Normally, someone would expect a low conservation of amino acids in coil. However, our conservation analysis showed a conservation of 71/76 for the WT and a conversation of 0/76 for our mutant W408 in our MSA of homologous mammalian sequences (see \citealp{msahomo} for the complete MSA). This might support the hypothesis that our WT amino acid is important for the coordination of these two domains. 

\paragraph*{Structure Based:}

%\begin{figure}[!tpb]
%\centerline{\includegraphics[width=0.5\textwidth]{408_int.png}}
%\caption{A visualization of the key players in the analysis of the mutation R408W. Red: residue 408; Blue: oligomerization domain; Dark-orange: bound iron; Magenta: BH4; Black: hbonds between the oligomerization domain and the catalytic domain}\label{fig:04}
%\end{figure}

The RMSF calculated by our molecular dynamics simulation for the WT of the structure identify the coil with the residue 408 to be flexible. This is probably a "hinge" loop between the oligomerization and the catalytic domain of the protein (see figure \ref{fig:allmutations}d). There are only five H-bonds between these two domains and three of them are defined by R408 (see table \ref{Tab:02}). The mutation causes several clashes in the prediction of SCWRL and probably these three H-bonds are broken. Therefore the flexibility between the two domains is somehow changed. PAH exists in the cell in an equilibrium between its homodimer and its homotetramer form. The dimer is less active than the tetramer form. It is supposed there is a mechanism, which changes the positions of the two domains relative to each other in order to realize this equilibrium. Through the dramatic change of the H-bonds between these two domains, this mechanism is probably broken, perhaps oligomerization itself is at least to some part broken. Therefore R408W is probably a dramatic change, which results in a significant decrease of PAH activity.

\section{Conclusion}
%Manual > Automatic Methods
We selected several known mutations of PAH. Some of them cause PKU others
do not. Automatic and manual approaches were used to determine the effect
of these mutations on PAH. We were interested, if it is possible to
distinguish the effect of a mutation with these methods. In this paper we
present only the selected PKU causing mutations, because they are more
interesting and they represent our conclusion well.
SNAP, minmise, FoldX are just some of the automatic methods. We also ran these
methods on our selected mutations of PAH, but the results were not
very significant. The more manual techniques like the analysis of the
hydrogen bonds and the literature search for PAH were much more
meaningful. While using the literature search we got more insight to the proteins
characteristics and dynamics. This insight allowed us to determine the
effect of most mutations. Therefore the introduction was larger to contain
this insight used by our conclusions. The automatic methods might have
their right to exist for a preselection of harmful mutations, but a very
coarse-grained preselection.

%Structure > Sequence Based
The basis of some of the used methods is just the sequence. These methods
were often quite insignificant. But they are sometimes the only methods
applicable for the analysis of mutations of unknown structures. Some of
these unresolved proteins can be approached by homology modeling, but for
proteins, that can not be modeled, these methods are the only tools
usable. Furthermore, we experienced that for sequence based conservation analysis the sequence search and selection step are more crucial than the actual method used for creating the multiple sequence alignment. We compared the conservation of our mutations once with homological sequences retrieved from HSSP and once with a BLAST search. We found out that the sequences from BLAST used for conservation analysis had a higher conservation for the mutations tested for than the sequences used from HSSP. This might cause false assumptions. 
The structure-based methods were much more useful. The effect of a
mutation in a protein might cause major three-dimensional changes. Without
a structure these changes are often not predictable.

%endg?ltige zusammenfassung
But even the structure-based methods and our knowledge-based conclusions
are just predictions. For a proper analysis of the mutations the structure
of the mutated protein might be useful. In the case of PAH a complete
structure of the native uncomplexed protein, the native tetra-homo-mere
and the native di-homo-mere would also be very valuable for further
analysis.

\newpage
\begin{thebibliography}{}
\bibitem[Erlandsen {\it et~al}., 2000]{Erlandsen} Heidi Erlandsen,H., Stevens,R.C. (1999) The Structural Basis of Phenylketonuria, {\it Molecular Genetics and Metabolism}, {\bf 68}, 103-125.

\bibitem[DiLella {\it et~al}., 1986]{DiLella} DiLella, A. G., Kwok, S. C. M., Ledley, F. D., Marvit, J., Woo, S. L. C. (1986) Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene, {\it Biochemistry}, {\bf 25}, 743-749.

\bibitem{folling1934ausscheidung}A.~Foelling.\newblock Ueber ausscheidung von phenylbrenztraubensaeure in den harnals stoffwechselanomalie in verbindung mit imbezillitaet. \newblock {\em Hoppe-Seyler{\'{}} s Zeitschrift fuer physiologische Chemie}, 227(1-4):169--181, 1934.

\bibitem{Guldberg01121995} P~Guldberg, K~F Henriksen, I~Sipil?, F~G?ttler, and A~de~la Chapelle. \newblock Phenylketonuria in a low incidence population: molecular characterisation of mutations in finland. \newblock {\em Journal of Medical Genetics}, 32(12):976--978, 1995.

\bibitem{blau2010phenylketonuria} N.~Blau, F.J. van Spronsen, and H.L. Levy. \newblock Phenylketonuria. \newblock {\em The Lancet}, 376(9750):1417--1427, 2010.

\bibitem{van2010phenylketonuria} F.J. van Spronsen. \newblock Phenylketonuria: a 21st century perspective. \newblock {\em Nature Reviews Endocrinology}, 6(9):509--514, 2010.

\bibitem{ozalp2001newborn} I.~Ozalp, T.~Co{\c{s}}kun, A.~Tokatli, HS~Kalkano{\u{g}}lu, A.~Dursun, S.~Tokol, G.~Koeksal, M.~Ozguec, and R.~Koese. \newblock Newborn pku screening in turkey: at present and organization for future. \newblock {\em The Turkish journal of pediatrics}, 43(2):97, 2001.

\bibitem{jiang2003survey} J.~Jiang, X.~Ma, X.~Huang, X.~Pei, H.~Liu, Z.~Tan, and L.~Zhu. \newblock A survey for the incidence of phenylketonuria in guangdong, china. \newblock {\em The Southeast Asian journal of tropical medicine and public health}, 34:185, 2003.
  
\bibitem{zhan2009neonatal} J.Y. Zhan, Y.F. Qin, and Z.Y. Zhao. \newblock Neonatal screening for congenital hypothyroidism and phenylketonuria in china. \newblock {\em World Journal of Pediatrics}, 5(2):136--139, 2009.

\bibitem{pangkanon2009detection} S.~Pangkanon, W.~Charoensiriwatana, N.~Janejai, W.~Boonwanich, and S.~Chaisomchit. \newblock Detection of phenylketonuria by the newborn screening program in thailand. \newblock {\em Southeast Asian J Trop Med Public Health}, 40:525--29, 2009.

\bibitem{guthrie1963simple} R.~Guthrie and A.~Susi. \newblock A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. \newblock {\em Pediatrics}, 32(3):338, 1963.

\bibitem{macdonald2007modern} M.J.M.D.M.J. MacDonald, BD~Godwin, and G.B.D.C. Cunha. \newblock A modern view of phenylalanine ammonia lyase. \newblock {\em Biochemistry and cell biology}, 85(3):273--282, 2007.

\bibitem{sarkissian2008preclinical} C.N. Sarkissian, A.~G{\'a}mez, L.~Wang, M.~Charbonneau, P.~Fitzpatrick, J.F. Lemontt, B.~Zhao, M.~Vellard, S.M. Bell, C.~Henschell, et~al. \newblock Preclinical evaluation of multiple species of pegylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria. \newblock {\em Proceedings of the National Academy of Sciences}, 105(52):20894, 2008.

\bibitem{jung2008protective} S.C. Jung, J.W. Park, H.J. Oh, J.O. Choi, K.I. Seo, E.S. Park, and H.Y. Park. \newblock Protective effect of recombinant adeno-associated virus 2/8-mediated gene therapy from the maternal hyperphenylalaninemia in offsprings of a mouse model of phenylketonuria. \newblock {\em Journal of Korean medical science}, 23(5):877, 2008.

\bibitem{rebuffat2010comparison} A.~Rebuffat, C.O. Harding, Z.~Ding, and B.~Th{\\"o}ny. \newblock Comparison of adeno-associated virus pseudotype 1, 2, and 8 vectors administered by intramuscular injection in the treatment of murine phenylketonuria. \newblock {\em Human gene therapy}, 21(4):463--477, 2010.

\bibitem{ding2008correction} Z.~Ding, C.O. Harding, A.~Rebuffat, L.~Elzaouk, J.A. Wolff, and B.~Th{\\"o}ny. \newblock Correction of murine pku following aav-mediated intramuscular expression of a complete phenylalanine hydroxylating system. \newblock {\em Molecular Therapy}, 16(4):673--681, 2008.

\bibitem{james2006andrews} W.D. James, T.G. Berger, D.M. Elston, and R.B. Odom. \newblock {\em Andrews' diseases of the skin: clinical dermatology}. \newblock Saunders Elsevier, 2006.

\bibitem{ensemblpah} http://asia.ensembl.org/Homo\_sapiens/Transcript/Summary?db=core;g=
ENSG00000171759;r=12:103291562-103331199;t=ENST00000307000 ;
\newblock last retrieved on 22nd of September 2011.

\bibitem{scriver2007pah} C.R. Scriver. \newblock The pah gene, phenylketonuria, and a paradigm shift. \newblock {\em Human mutation}, 28(9):831--845, 2007.

\bibitem{listpahstruc} http://www.uniprot.org/uniprot/P00439\#section\_x-ref ; \newblock last retrieved on 22nd of September 2011.

\bibitem{andersen2001high} O.A. Andersen, T.~Flatmark, and E.~Hough. \newblock High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active fe (ii) form and binary complex with tetrahydrobiopterin1. \newblock {\em Journal of Molecular Biology}, 314(2):279--291, 2001.

\bibitem{pdb1j8u} http://www.rcsb.org/pdb/explore/explore.do?structureId=1J8U ; last retrieved on 22nd of September 2011.

\bibitem{kabsch1983dictionary} W.~Kabsch and C.~Sander. \newblock Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. \newblock {\em Biopolymers}, 22(12):2577--2637, 1983.

\bibitem{jones1999protein} D.T. Jones. \newblock Protein secondary structure prediction based on position-specific scoring matrices1. \newblock {\em Journal of molecular biology}, 292(2):195--202, 1999.

\bibitem{canutescu2003graph} A.A. Canutescu, A.A. Shelenkov, and R.L. Dunbrack~Jr. \newblock A graph-theory algorithm for rapid protein side-chain prediction. \newblock {\em Protein science}, 12(9):2001--2014, 2003.

\bibitem{poirot20043dcoffee} O.~Poirot, K.~Suhre, C.~Abergel, E.~O'Toole, and C.~Notredame. \newblock 3dcoffee@ igs: a web server for combining sequences and structures into a multiple sequence alignment. \newblock {\em Nucleic acids research}, 32(suppl 2):W37, 2004.

\bibitem{dayhoff1978model} M.O. Dayhoff and R.M. Schwartz. \newblock A model of evolutionary change in proteins. \newblock In {\em In Atlas of protein sequence and structure}. Citeseer, 1978.

\bibitem{henikoff1992amino} S.~Henikoff and J.G. Henikoff. \newblock Amino acid substitution matrices from protein blocks. \newblock {\em Proceedings of the National Academy of Sciences}, 89(22):10915, 1992.

\bibitem{altschul1997gapped} S.F. Altschul, T.L. Madden, A.A. Sch{\\"a}ffer, J.~Zhang, Z.~Zhang, W.~Miller, and D.J. Lipman. \newblock Gapped blast and psi-blast: a new generation of protein database search programs. \newblock {\em Nucleic acids research}, 25(17):3389, 1997.

\bibitem{schymkowitz2005foldx} J.~Schymkowitz, J.~Borg, F.~Stricher, R.~Nys, F.~Rousseau, and L.~Serrano. \newblock The foldx web server: an online force field. \newblock {\em Nucleic acids research}, 33(suppl 2):W382, 2005.

\bibitem{van2005gromacs} D.~Van Der~Spoel, E.~Lindahl, B.~Hess, G.~Groenhof, A.E. Mark, and H.J.C. Berendsen. \newblock Gromacs: fast, flexible, and free. \newblock {\em Journal of computational chemistry}, 26(16):1701, 2005. 

\bibitem{pymol} http://www.pymol.org/ ; last retreived 29th of September 2011.

\bibitem{fitzpatrick2003} P.F. Fitzpatrick. \newblock Mechanism of aromatic amino acid hydroxylation. \newblock {\em Biochemistry}, 42(48), 2003.

\bibitem{fitzpatrick2010} J. Li, L.J. Dangott and P.F. Fitzpatrick. \newblock Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry. \newblock {\em Biochemistry}, 49(15), 2010.

\bibitem{blossummatrix} http://goo.gl/f6jlY ; last retrieved on 30th of September 2011.

\bibitem{pammatrix} http://goo.gl/850Kz ; last retrieved on 30th of September 2011.

\bibitem{pssmmatrix} http://goo.gl/uclEH ; last retrieved on 30th of September 2011.

\bibitem{msahomo} http://goo.gl/1WmsQ ; last retrieved on 30th of September 2011.

\bibitem{fitzpatrick2011} J. Li, U. Ilangovan , S.C. Daubner, A.P. Hinck and P.F. Fitzpatrick. \newblock Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase. \newblock {\em Arch Biochem Biophys}, 505(2), 2011.

\bibitem{Kobe1999} B. Kobe, I.G. Jennings, C.M. House, B.J. Michell, K.E. Goodwill, B.D. Santarsiero, R.C. Stevens, R.G. Cotton and B.E. Kemp \newblock Structural basis of autoregulation of phenylalanine hydroxylase  \newblock {\em Nat. Struct. Biol.}, 5(5), 1999.

\bibitem{Bassan2003} A. Bassan, M.R. Blomberg and P.E. Siegbahn \newblock Mechanism of dioxygen cleavage in tetrahydrobiopterin-dependent amino acid hydroxylases. \newblock {\em Chemistry}, 9(1), 2003.

\bibitem{Olsson2011} E. Olsson, A. Martinez, K. Teigen and V.R. Jensen VR \newblock Formation of the iron-oxo hydroxylating species in the catalytic cycle of aromatic amino acid hydroxylases. \newblock {\em Chemistry}, 17(13), 2011.

\bibitem{Flatmark1999} T. Flatmark and R.C. Stevens \newblock Structural Insight into the Aromatic Amino Acid Hydroxylases and Their Disease-Related Mutant Forms. \newblock {\em Chem. Rev.}, 99(8), 1999.

\bibitem{Bjorgo2001} E. Bj\o rgo, R.M. de Carvalho, T. Flatmark \newblock A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427$-->$Pro mutant human phenylalanine hydroxylase: contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding. \newblock {\em Eur. J. Biochem}, 268(4), 2001.

\bibitem{nick1998helix} C.~Nick~Pace and J.~Martin~Scholtz. \newblock A helix propensity scale based on experimental studies of peptides and proteins. \newblock {\em Biophysical journal}, 75(1):422--427, 1998.

\end{thebibliography}




\end{document}
